The application proposes to identify cellular and molecular components that contribute to the differentiation and spatial organization of tissues in the early vertebrate embryo. One avenue that has proven particularly informative for elucidating how individual cell fates and overall body plan are determined in invertebrates has been the analysis of developmental mutations. Recent studies with the zebrafish (Brachydanio rerio) have proven that this vertebrate has many of the attributes required for developmental genetic studies. The longterm goal of the study is to perform a detailed genetic and molecular characterization of genes that contribute to early development and that lie in either of two regions of the genome defined by the albino and yellow loci. The goal of this application is to establish a series of overlapping deletion mutations near the albino or yellow loci. Heritable deficiency mutations will be induced following gamma irradiation of developing zebrafish embryos. A total of 500 mutagenized fish will be mated with individuals harboring recessive, viable, pigmentation mutations at albino or yellow to identify the transmission of newly induced, non-complementing mutations. Approximately 1 percent of the mutagenized fish are expected to harbor a new mutation at each pre-specified locus. Each of the induced mutations will be characterized genetically. The functions perturbed by the mutations will be analyzed by examination of the phenotypes of homozygous mutant embryos. Pairwise complementation tests between each of the induced mutations will: 1) identify deletion mutations that affect several gene functions; 2) help determine the minimal number of distinct gene functions affected by each series of recessive lethal mutations; and 3) permit construction of deficiency maps for the albino and yellow regions of the genome. To demonstrate directly that some of the rat-induced mutations are deletions, homozygous mutants will be analyzed for loss of any of 50 specific DNA sequence loci. The proposed project represents an efficient approach to the recovery of developmental mutants in the zebrafish.The deletion mutations will represent a first step toward complete genetic and molecular characterization of the genes near albino and yellow that regulate early development. Analysis of the developmental defects associated with these mutations will reveal cellular and molecular functions that are essential to early vertebrate embryogenesis.